Detection of the transcripts of STAT6(A) and STAT6(B) by Northern blot. Filters (Clontech) containing preblotted mRNA (2 μg of each) from different adult human tissues were separately hybridized with an α-³²P-dCTP-labeled DNA probe of STAT6(A) (full-length) or STAT6(B) (amino acids 1–150) and then autoradiographed with Biomax MR film (Kodak).

Detection of LPS-induced LITAF, STAT6(A), or STAT6(B) production in THP-1 cells. Cells were untreated (lane 1) or pretreated with LPS at 5 (lane 2), 25 (lane 3), 50 (lane 4), 100 (lane 5), or 200 (lane 6) ng/ml for 3 h, then washed with PBS and maintained overnight. Cell protein lysates were extracted and subjected to Western blot analyses.

Detection of translocation of LITAF-STAT6(B) complex in THP-1 cells. Protein extracts were collected from whole cells, cytoplasm, or nuclei after overexpression of both LITAF and STAT6(B). The alterations in the protein levels of exogenous LITAF or STAT6(B) and endogenous STAT6(A) over time were measured by Western blot with the following antibodies: LITAF (611615, BD Biosciences), Stat6 (S-20, Santa Cruz Biotechnology), or β-tubulin (C-20, Santa Cruz Biotechnology) as control.

Detection of shRNA-silenced LITAF gene expression. (A) Ten micrograms of total RNA extracts from LPS-stimulated THP-1 cells after transfection of 0 (lanes 1 and 2), 0.25 (lane 3), or 0.5 (lane 4) μg of pSHAG-LITAF-DNA to induce shRNA were isolated and subjected to Northern blot analysis with an α-³²P-dCTP-labeled DNA probe of LITAF (full-length) or GAPDH (Clontech). (B) The protein extracts from the treated cells were probed with the following antibodies: LITAF (611615, BD Biosciences) or actin (C-11, Santa Cruz Biotechnology) as a constitutive control. (C) Transcriptional activity of a series of deletion constructs of TNF-α promoter DNA. U2OS cells were transiently transfected with LITAF alone, STAT6(B) alone, or empty vector (mock) as control or were cotransfected with LITAF plus pSHAG-LITAF, LITAF plus STAT6(B), or LITAF plus STAT6(B) plus pSHAG-LITAF. Subsequent luciferase production was measured as described (9) in triplicate assays. The β-gal gene was included in all transfections. The promoter activity of each test sample was normalized to β-gal in the same lysates as described (15) and graphed. (D) ELISA in THP-1 cells. Cells were transiently transfected with LITAF alone, STAT6(A) alone, STAT6(B) alone, or empty vector as control and cotransfected with LITAF plus STAT6(A), LITAF plus STAT6(B), LITAF plus pSHAG-LITAF, or LITAF plus STAT6(B) plus pSHAG-LITAF. After 16 h of incubation, supernatants from treated cells were collected, and TNF-α secreted from each was measured by ELISA, in triplicate assays. The β-gal gene was included in all transfections. The density value of each test sample was normalized to β-gal in the same lysates and graphed.

Immunoprecipitation of murine macrophages. The cell lysates from untreated (lanes 1, 3, and 5) or LPS-treated (100 ng/ml LPS) (lanes 2, 4, and 6) macrophages were immunoprecipitated with anti-LITAF (lanes 3 and 4) or anti-STAT6 (lanes 5 and 6), and then protein A/G beads (SC-2003, Santa Cruz Biotechnology) were added. The precipitates were subjected to Western blotting with the antibodies indicated by arrows.

EMSA of the protein–DNA interaction. Protein (30 μg) extracted from each culture of transfected U2OS cells was added to the appropriate reaction buffer with the oligonucleotide DNA probe, ³²P-ATP-labeled hTNF-α promoter DNA as described (9). Protein from untransfected cells was applied in lanes 1 and 2, and a 50-fold excess of unlabeled competitor was added (lane 2). The proteins from each condition of cells treated with 1 μg of DNA of empty vector alone (lane 3), full-length LITAF alone (lane 4), both full-length LITAF and STAT6(A) (lane 5), full-length LITAF plus a fragment of STAT6(B) (amino acids 1–150, lane 6), full-length LITAF plus a fragment of STAT6(B) (amino acids 151–404, lane 7), and full-length LITAF plus full-length STAT6(B) (amino acids 1–404, lane 8) were assessed by EMSA. The shifted DNA bands are indicated by arrows.

Cytokine antibody array and Western blot of DNA-transfected THP-1 cells. (A) Human 44 cytokines were blotted onto a membrane and arrayed three times following the manufacturer's protocol. The intensities of the relative expression levels of cytokines were quantified by densitometry (VersaDoc imaging system, Bio-Rad). The β-gal gene was included in all transfections. The density value of each test sample was normalized to β-gal from the same lysates as described (15) and graphed. (B) Detection of LITAF/STAT6(B) complex-regulated gene expression in THP-1 cells. Proteins from THP-1 whole cells without DNA transfection (lane 1) or after DNA transfection with empty vector (lane 2), overexpression of LITAF alone (lane 3), STAT6(A) alone (lane 4), STAT6(B) alone (lane 5), and both LITAF and STAT6(B) (lane 6) were extracted and subjected to Western blot analyses. A total of 80 μg of protein extract was loaded per lane. Blots were probed with the following antibodies: Stat6 (S-20, Santa Cruz Biotechnology), LITAF (611615, BD Biosciences), IL-1α (R-20, Santa Cruz Biotechnology), IL-10 (M-18, Santa Cruz Biotechnology), MCP-2 (C-17, Santa Cruz Biotechnology), RANTES (C-19, Santa Cruz Biotechnology), or actin (C-11, Santa Cruz Biotechnology) as control.